Method for the qualification of preparations of pentosan polysulfate, raw materials and production processes thereof

ABSTRACT

A method for the qualification and selection of manufacturing processes, raw materials, intermediates and batch production of pentosan polysulfate based on the identification of acetylated monosaccharide units, including units of xylose substituted with 4-O-methyl-glucuronic which also lead the acetyl group, as structural characterizing units, is disclosed.

This application is a Divisional Application of U.S. Ser. No. 14/762,784filed on Jul. 22, 2015, which is a U.S. National Stage ofPCT/EP2014/051347 filed 23 Jan. 2014 which claims priority to and thebenefit of Italian Application No. MI2013A000112 filed 24 Jan. 2013, thecontents of which are incorporated herein by reference in theirentireties.

The present invention relates to methods and products based on theidentification of characterizing structural units of pentosanpolysulfate and the corresponding xylan without sulfate groups, inparticular to products obtained by a method of qualification andselection of production processes, raw materials, intermediates,production batches of pentosan polysulfate based on the identificationof said characterizing structural units.

STATE OF THE ART

Several pharmaceutical products (for example Elmiron®, CCRIS 8869,Fibrase®, Fibrezym®, HSDB 7294, Hoe/bay 946, NSC 626201, SP-54,Thrombocid®, PZ68, H_(emoc)l_(ar)®, Fibrocid® and Tavan®) contain, asactive ingredient, pentosan polysulfate, a polysaccharide which mainskeleton is constituted by sequences of sulfated xylose units linkedtogether through a glycosidic β-(1→4) bond.

Being obtained from xylanes extracted by high trees such as beech,ramifications of 4-O-methyl-glucuronic acid are present and distributedin a not necessarily regular manner.

The chemical structure of pentosan polysulfate reported in the technicaland scientific literature corresponds to the following formula 1:

wherein R is H or SO₃Na.

The commercial product Elmiron® has a high sulfation degree and itsmolecular weight is 4000-6000 Dalton. Therefore pentosan polysulfate ismade of a mixture of sulfated polysaccharides different each other forchain lengths and branching.

Pentosan polysulfate is used in the pharmaceutical field asanticoagulant and in the treatment of interstitial cystitis. It alsoshows several other biological activities including anti-tumor andanti-metastatic activity, antiviral and anti-inflammatory activity. Ithas been proposed as a therapeutic agent in the prevention and treatmentof Prion Disease. It has effect in inhibiting the growth of the calciumoxalate crystals which lead to the formation of kidney stones. It alsoshowed efficacy as anti-arthritic agent in animals suffering fromosteoarthritis.

The polymerization degree and the structural variety can stronglyinfluence the biological, immunological and toxicological activity ofpolysaccharides and of sulfated polysaccharides [T. Astrup Scand J ClinLab Invest 137 (1952)].

A deep knowledge of the structural characteristics of pentosanpolysulfate is therefore extremely important to ensure the therapeuticefficacy and safety of the pharmacological treatments which use it.

Because of the natural origin of the raw material and because of theprocesses of isolation and production, structural groupings whichcontribute to increase its structural complexity may be present on thepolymer chain of xylan polysulfate. For example, in addition to sulfatedxylose and sulfated 4-O-methyl-glucuronic acid, different monosaccharideunits may be present.

Furthermore different sulfated and not sulfated polymeric species may bepresent, such as for example those deriving from other polysaccharidespresent in the natural source of the raw material.

In particular, the structural complexity of pentosan polysulfate mayfurther increase depending on the method for the extraction of xylanfrom the natural source of the raw material. In fact such extractionmethod, depending from its selectivity, will provide xylan withdifferent characteristics and different purity degrees. Moreover, duringthe transformation of xylan into the final product, it is possible thatnew impurities are introduced and that the transformation of thosepresent or the introduction of new chemical groups in the polymerstructure occur. These factors may also contribute to the complexity ofthe product as a whole.

Currently, the structural characterization of pentosan polysulfate hasnot been brought to the level of details required in view of thecomplexity of the product and therefore it is not suitable to the needof ensuring quality standard and equivalence of the production of thedrugs on the market and to the proof of equivalence of genericpharmaceutical products.

A progress in the analytical characterization of pentosan polysulfateand in the identification of its characteristic structural units istherefore urgently needed.

In the present context, characteristic structural unit means a part ofthe chemical structure of pentosan polysulfate or of xylan used for itspreparation. Based on said characteristic structural unit variouscompositions or preparation methods of these polysaccharides can bequalified.

Qualification means the acknowledgement of the suitability of apolysaccharide composition or of a method for its preparation for themanufacturing of a pharmaceutical product for human use.

SUMMARY OF THE INVENTION

The invention is based on the discovery that the structure of pentosanpolysulfate in the pharmaceutical products is characterized by thepresence of some characteristic structural units that have not been sofar identified in the chemical structure of formula 1 reported in theliterature.

Such characteristic structural units have been identified by theApplicant and are acetylated monosaccharide units, and in particularunits of acetylated xylose substituted with 4-O-methyl-glucuronic acid.

Therefore the present invention relates to methods for identifying thepresence of said characteristic structural units in pentosan polysulfateand in xylan and to their application for the qualification ofpreparations of pentosan polysulfate, their raw materials such as xylanand their production processes.

A further object of the present invention is a method for thepreparation of pentosan polysulfate and xylan comprising at least onequalification step based on said characteristic structural units.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1—¹H-NMR spectrum of pentosan polysulfate

FIG. 2—(A) HSQC NMR spectrum of pentosan polysulfate (XS) and (B) ofxylan without sulfate groups obtained by desulfation of pentosanpolysulfate (XSDS)

FIG. 3—Maldi TOF spectrum of xylan XSDS

DETAILED DESCRIPTION OF THE INVENTION

In a first aspect the present invention relates to polysaccharides offormula 2

wherein

R is H or SO₃Na;

G is H, SO₃Na or acetyl;

obtained by a method comprising at least one identification orquantification step of acetylated monosaccharide units, and inparticular units of acetylated xylose substituted with4-O-methyl-glucuronic acid.

Pentosan polysulfate of formula 2 is characterized by the presence ofxylose units substituted with 4-O-methyl-glucuronic acid and acetylated(G=acetyl) which represent characteristic structural units identified bythe Applicant.

In a further aspect, the present invention relates to methods foridentifying the presence of said characteristic structural units inpentosan polysulfate as well as in xylan and to their application forthe qualification of preparations of pentosan polysulfate, their rawmaterials (xylans) and their production processes.

In a further embodiment, the present invention relates to the xylan offormula 2 wherein R is hydrogen and G is hydrogen or acetylcharacterized by the presence of xylose units substituted with4-O-methyl-glucuronic acid and acetylated (G=acetyl) which representcharacteristic structural units identified by the Applicant.

The invention is based on the discovery that the structure of pentosanpolysulfate present in pharmaceutical products is characterized by thepresence of some characteristic structural units which may be correlatedto the therapeutic efficacy and safety of pentosan polysulfate.

In the present context, the terms “pentosan polysulfate forpharmaceutical use”, “pharmaceutical products containing pentosanpolysulfate” and “commercial pentosan polysulfate” mean products on themarket and/or known products which contain pentosan polysulfate asactive ingredient for pharmaceutical use, with preferred reference tothe medicinal product marketed under the name Elmiron®.

By analyzing samples of commercial pentosan polysulfate by ¹H-NMRspectroscopy, in particular Elmiron®, the signals corresponding to thepolysulfate structure with high sulfation degree of formula 1 whereinthe R groups are mainly sulfate groups have been detected, andfurthermore it has been unexpectedly found that the spectrum shows thepresence of signals at about 2.3 ppm corresponding to the presence ofacetyl groups and the splitting of some peaks. In particular thesplitting of the signal at about 3.48 ppm corresponding to the methoxygroup of the 4-O-methyl-glucuronic acid units stands out (FIG. 1).Signals in the region 8.8-9.3 ppm due to pyridinium groups arising fromthe sulfation procedure used for the production of pentosan polysulfatefor pharmaceutical use have been also detected. The presence ofpyridinium groups bound to pentosan polysulfate is reported in theliterature, for example in WO03073106 and Carbohydrate Polymers 16(1991) 211-214.

On the contrary, the splitting of the ¹H-NMR signal relative to themethoxy group of the unit of 4-O-methyl-glucuronic acid and the presenceof signals corresponding to acetyl groups are not expected on the basisof the chemical structure of pentosan polysulfate reported in thetechnical and scientific literature and represented by formula 1.

The Applicant has taken into consideration the possibility that the NMRsignal at about 2.3 ppm was caused by traces of solvents or salts usedin the production process of pentosan polysulfate and not completelyremoved during purification.

Chemical analyses such as the determination of the free anions by HPLC,gas chromatographic analysis of residual solvents and comparison of the¹H-NMR chemical shifts in the 2.3 ppm region allowed to exclude thepresence of species such as acetate ion and solvents such as acetic acidand ethyl acetate in such quantities to justify the presence of the¹H-NMR signal at about 2.3 ppm in the spectrum of pentosan polysulfateshown in FIG. 1.

Said ¹H-NMR signal was then attributed to the presence of an acetylgroup chemically bound to pentosan polysulfate.

The xylan from beech is used as a raw material in the manufacture ofpharmaceutical products of pentosan polysulfate for human use.

The native xylan from beech is constituted by chains of beta 1-4 xylosepartially acetylated at positions 2 and 3; there are also units of alpha4-O-methyl-glucuronic acid bound to position 2 of the xylose unit of thepolysaccharide chain [see for example Carbohydrate Research 337 (2002)373-377]. The processes commonly used for the isolation of xylan frombeech involve the use of strongly alkaline solutions in which the xylanshows a quite good solubility; under these conditions the completedeacetylation of xylan occurs.

It is therefore surprising to have identified the presence of the acetylfunction bound to pentosan polysulfate; in view of the potentialimplications on the biological and toxicological activities of pentosanpolysulfate for pharmaceutical use, the acetyl group is a characterisingstructural units of the product.

For the structural identification of the unit characterizing pentosanpolysulfate, experiments based on NMR spectroscopy were carried outexploiting the potential of this technique in the structural analysis ofpolysaccharides and sulfated polysaccharides.

The NMR spectrum of Heteronuclear Single Quantum Coherence (HSQC) ofsamples of pentosan polysulfate shows a series of signals correspondingto the different positions of the units of xylose and4-O-methyl-glucuronic acid of the polysaccharide (FIG. 2-A).

The attribution of each detected signals to specific chemically definedpositions is made difficult by the large number of structuralcombinations similar to each other present in the polysaccharide, andtherefore by the presence of a multitude of signals in the HSQC NMRspectrum; these signals are close each other in the spectrum andpartially overlapped. The multiplicity of positions chemically similar,but spectroscopically distinguishable derives from the combination ofthe various possible patterns of substitution with functional groups(sulfate, hydrogen or acetyl) or ramifications of the xylose unit of thechain, reducing end or non-reducing end. The signals corresponding tothe units of 4-O-methyl-glucuronic acid which are present in the variouspossible structural variety overlap to the signals corresponding to theabove reported species.

This situation hampers the work of attribution of all the HSQC NMRsignals to specific structural contexts.

By repeating the NMR experiment on samples of pentosan polysulfate forpharmaceutical use that had been subjected to a procedure of exhaustivedesulfation to obtain the corresponding xylan without sulfate groups, ahigher resolution of the signals and the simplification of the HSQCspectrum was found. The chemical environments corresponding to thedifferent positions of the polysaccharide are apparently moredifferentiated each other in the desulfated product than in the sulfatedproduct. In addition, the removal of the sulfate groups reduces thestructural variability of the different constituent units of thepolysaccharide taking into consideration that also in pentosanpolysulfate, notwithstanding its high sulfation degree, not completelysulfated saccharide units are present and that each of these can beassociated with a series of NMR signals.

Therefore a reduction of the structural variety corresponds to asimplification of the NMR spectrum (FIG. 2-B).

Therefore it was possible, by combining several NMR techniques such asanalysis HSQC, HMBC, TOCSY and COSY, to find that the acetyl group isnot equally distributed between the positions 2 and 3 of the repeatingunits which constitute pentosan polysulfate, but it is mainly present atposition 3 of the repeating units of xylose.

Furthermore the Applicant found that said acetyl groups are mainly boundto the position 3 of the xylose units which have also a4-O-methyl-glucuronic acid unit in position 2.

The Applicant has therefore determined experimentally that pentosanpolysulfate for pharmaceutical use is characterized by the presence ofunits of xylose substituted with 4-O-methyl-glucuronic acid andacetylated which represent structural characteristic units.

This determination of the structural units of the polysaccharide hasbeen confirmed by the mass spectroscopy analysis of the xylan obtainedby exhaustive desulfation of pentosan polysulfate on which a Maldi TOFanalysis was carried out. FIG. 3 shows the resultant spectrum showingthe presence of three different sets of sequences that differ by thevalue corresponding to the mass of the repeating unit of xylose (132daltons), having the following masses:

-   -   the first series having peaks with mass of 701, 833, 965, 1165        Da, etc. corresponding to oligomers of polyxylan (Xyl)n    -   the second series having peaks with mass of 759, 891, 1023, 1155        Da, etc. corresponding to oligomers of polyxylan with a branch        of 4-O-methyl-glucuronic acid (Xyl)n-(Xyl-GlcAOMe)-(Xyl)m    -   the third series having peaks with mass of 801, 933, 1065 Da,        etc. corresponding to oligomers of polyxylan with a branch of        4-O-methyl-glucuronic acid in which an acetyl group is also        present (Xyl)n-(Xyl-OAc-GlcAOMe)-(Xyl)m

The results obtained from the characterization by MALDI TOF massspectroscopy show that the acetyl group is bound to the structure of thepolysaccharide. Said results also show that the acetyl group is mainlypresent in species in which there is also a 4-O-methyl-glucuronic acidgroup. By suitably combining the results of the NMR analysis it waspossible to fill in the following Table 1 with the assignment of the NMRsignals relative to the sample obtained by desulfation procedure.

TABLE 1 Sac- Posi- cha- ¹H ¹³C tion ride Type ppm ppm 1 Xyl internal4.47 104.61 Xyl nr 4.44 104.87 Xyl redα 5.17 94.98 Xyl redβ 4.57 99.47Xyl having 4-MGA bound in 2 4.74 103.61 Xyl acetylated in 3 + 4-MGAbound in 2 4.63 104.17 4-MGA bound to Xyl 5.27 100.88 4-MGA bound to Xylacetylated in 3 5.30 101.16 2 Xyl internal 3.29 75.69 Xyl nr 3.29 75.69Xyl redα 3.53 74.39 Xyl redβ 3.24 77.00 Xyl having 4-MGA bound in 2 Xylacetylated in 3 + 4-MGA bound in 2 3.68 78.41 4-MGA bound to Xyl 3.5873.93 4-MGA bound to Xyl acetylated in 3 3.58 73.93 3 Xyl internal 3.5576.68 Xyl nr 3.42 78.66 Xyl redα 3.75 74.00 Xyl redβ 3.55 76.68 Xylhaving 4-MGA bound in 2 Xyl acetylated in 3 + 4-MGA bound in 2 5.0976.99 4-MGA bound to Xyl 3.65 75.21 4-MGA bound to Xyl acetylated in 33.80 75.22 4 Xyl internal 3.78 79.36 Xyl nr 3.62 72.22 Xyl redα 3.7879.36 Xyl redβ 3.78 79.36 Xyl having 4-MGA bound in 2 3.78 79.36 Xylacetylated in 3 + 4-MGA bound in 2 3.94 78.87 4-MGA bound to Xyl 3.8084.31 4-MGA bound to Xyl acetylated in 3 3.82 84.46 5 Xyl internal 4.1065.96 Xyl nr 3.96 68.21 Xyl redα 3.81 61.86 Xyl redβ 4.10 65.96 Xylhaving 4-MGA bound in 2 4.10 65.96 Xyl acetylated in 3 + 4-MGA bound in2 4.10 65.96 4-MGA bound to Xyl 4.03 73.22 4-MGA bound to Xyl acetylatedin 3 4.64 72.67 5′ Xyl internal 3.37 65.95 Xyl nr 3.30 68.21 Xyl redα3.74 61.86 Xyl redβ 3.37 65.95 Xyl having 4-MGA bound in 2 3.37 65.95Xyl acetylated in 3 + 4-MGA bound in 2 3.37 65.95 Others 4-MGA O—CH₃bound to Xyl 3.49 63.00 4-MGA O—CH₃ bound to Xyl acetylated in 3 3.4963.00 4-MGA —COOH MGA bound to Xyl 176.50 4-MGA —COOH MGA bound to Xyl177.00 acetylated in 3 Xyl O—Ac (methyl) 2.17 23.89 Xyl O—Ac (carbonyl)176.60 TSP 0 Xyl—xylose 4-MGA—4-O-methyl-glucuronicTSP—trimethylsilylpropionate Ac—acetyl nr—saccharide unit at thenon-reducing end redα—saccharide unit at the reducing end αredβ—saccharide unit at the reducing end β

By using the information collected from the analysis of samples of xylanobtained by desulfation of pentosan polysulfate it was possible tocomplete the assignment of the NMR signals for the not desulfatedproduct. Combining this information with the results of the NMRexperiments, such as HSQC, HMBC, TOCSY and COSY, carried out directly onnot desulfated samples the results of the structural analysis wereconfirmed and the following table 2 has been prepared on the basis ofthe ¹H and ¹³C NMR signals of pentosan polysulfate.

TABLE 2 Sac- Posi- cha- ¹H ¹³C tion ride Type ppm ppm 1 Xyl(S) internal5.17 102.40 Xyl(S) nr 5.17 102.40 Xyl(S) redα 5.39 93.68 Xyl(S) redβ5.10 101.38 Xyl(S) having 4-MGA(S) bound to 2 Xyl(S) acetylated in 3 +4-MGA(S) 5.00 106.73 bound to 2 4-MGA(S) bound to Xyl(S) 5.79 96.864-MGA(S) bound to Xyl(S) acetylated in 3 5.76 96.99 2 Xyl(S) internal4.47 75.85 Xyl(S) nr 4.49 75.39 Xyl(S) redα 4.35 78.16 Xyl(S) redβ 4.4275.88 Xyl(S) having 4-MGA(S) bound to 2 Xyl(S) acetylated in 3 +4-MGA(S) 3.93 76.85 bound to 2 4-MGA(S) bound to Xyl(S) 4.32 78.224-MGA(S) bound to Xyl(S) acetylated in 3 4.31 77.76 3 Xyl(S) internal4.80 75.36 Xyl(S) nr 4.83 74.20 Xyl(S) redα 4.76 78.55 Xyl(S) redβ 4.7475.99 Xyl(S) having 4-MGA(S) bound to 2 Xyl(S) acetylated in 3 +4-MGA(S) 5.25 75.96 bound to 2 4-MGA(S) bound to Xyl(S) 4.95 80.044-MGA(S) bound to Xyl(S) acetylated in 3 4.42 80.83 4 Xyl(S) internal3.97 77.38 Xyl(S) nr 4.57 74.40 Xyl(S) redα 3.89 77.78 Xyl(S) redβ 3.9776.70 Xyl(S) having 4-MGA(S) bound to 2 Xyl(S) acetylated in 3 +4-MGA(S) 4.14 76.79 bound to 2 4-MGA(S) bound to Xyl(S) 3.45 84.044-MGA(S) bound to Xyl(S) acetylated in 3 3.45 84.04 5 Xyl(S) internal4.44 61.92 Xyl(S) nr 4.51 61.40 Xyl(S) redα 3.94 63.69 Xyl(S) redβ 4.4461.92 Xyl(S) having 4-MGA(S) bound to 2 Xyl(S) acetylated in 3 +4-MGA(S) 4.29 65.60 bound to 2 4-MGA(S) bound to Xyl(S) 4.36 75.054-MGA(S) bound to Xyl(S) acetylated in 3 3.92 75.71 5′ Xyl(S) internal3.86 61.92 Xyl(S) nr 3.89 61.40 Xyl(S) redα 3.94 63.64 Xyl(S) redβ 3.8661.92 Xyl(S) having 4-MGA(S) bound to 2 Xyl(S) acetylated in 3 +4-MGA(S) 3.69 65.60 bound to 2 Others 4-MGA(S) O—CH₃ bound to Xyl(S)3.50 63.37 4-MGA(S) O—CH₃ bound to Xyl(S) 3.48 63.61 acetylated in 34-MGA(S) —COOH 4-MGA(S) bound to 178.90 Xyl(S) 4-MGA(S) —COOH 4-MGA(S)bound to 178.44 Xyl(S) acetylated in 3 Xyl(S) O—Ac (methyl) 2.30 23.37Xyl(S) O—Ac (carbonyl) 176.14 Xyl(S)—xylose sulfate4-MGA(S)—4-O-methyl-glucuronic sulfate Ac—acetyl nr—saccharide unit atthe non-reducing end redα—saccharide unit at the reducing end αredβ—saccharide unit at the reducing end β

Based on the obtained results, the Applicant has attributed to pentosanpolysulfate for pharmaceutical use the structure of formula 2:

wherein

R is H or SO₃Na

G is H, SO₃Na or acetyl;

wherein the structural group G which can be an acetyl group, is present.

The Applicant has then experimentally defined that pentosan polysulfatefor pharmaceutical use is characterized by the presence of xylose unitssubstituted by 4-O-methyl-glucuronic acid and acetylated (G=acetyl) ascharacteristic structural units.

This high structural specificity is a characterizing feature of pentosanpolysulfate used as active pharmaceutical ingredient for human use.

The identification of this specific structural unit of pentosanpolysulfate has several useful applications such as the selection of themethod of isolation of the raw material (xylan from beech), theevaluation of the process for the transformation of xylan into the finalproduct comprising the steps of depolymerization and sulfation, theprocedures for the isolation and purification of the resultant pentosanpolysulfate, the evaluation of batches of the production ofintermediates and final product.

The above features has a further practical application of the inventionconsisting in the xylan obtained by the desulfation of pentosanpolysulfate or by other methods and represented by formula 2 wherein Ris hydrogen and G is hydrogen or acetyl as well as in the use of saidxylan as raw material for the preparation of pentosan polysulfate forpharmaceutical use by preserving its structural units.

A further practical embodiment of the present invention is thepossibility to make more meaningful the process controls at differentproduction steps by linking them to specific structural parameters.

These factors will be taken into account in the production and marketingof pentosan polysulfate for pharmaceutical use and should also beconsidered as part of the registration process of the product before theregulatory authorities, such as the FDA, for the authorization ofgeneric versions of pentosan polysulfate.

These assessments are not limited to the check of the presence of thecharacteristic structural unit, but also to its quantification. For thispurpose, NMR spectroscopy may still be used. Through this technique wehave found that the presence of the acetyl groups is mainly in theposition 3 of the repetitive xylose units. In addition we have foundthat said acetyl groups are mainly bound to the position 3 of xyloseunits also having a 4-O-methyl-glucuronic acid unit bound in position 2.By integration in the HSQC spectrum of different samples of xylan andpentosan polysulfate, the amount of xylose units having both an acetylgroup and a 4-O-methyl-glucuronic acid unit was determined (determinedby integrating the signal relative to the position 3 of the specifiedxylose unit), with respect to the total xylose units bearing a4-O-methyl-glucuronic acid unit in position 2 (integrating thecorresponding signals of the positions 1). Based on the obtained data,the Applicant has been able to determine that the content of xyloseunits substituted with 4-O-methyl-glucuronic acid which also have anacetyl group in the pentosan polysulfate for pharmaceutical use and inthe corresponding xylan without sulfate groups is at least 20%,preferably between 35% and 70%, with respect to the total amount of thepresent residues of 4-O-methyl-glucuronic acid.

Therefore, preferred features of the present invention are xylan andpentosan polysulfate of formula 2 containing at least 20% of xyloseunits substituted with a 4-O-methyl-glucuronic acid that are acetylatedas characteristic structural units obtained with a method that comprisesat least one qualification step based on said characteristic structuralunits.

In further particularly preferred aspects the present invention relatesto xylan and pentosan polysulfate of formula 2 containing between 35%and 70% of xylose units substituted with a 4-O-methyl-glucuronic acidthat are acetylated as characteristic structural units obtained with amethod that comprises at least one qualification step based on saidcharacteristic structural units.

This quantitative evaluation involves multiple advantages such as theselection of the method of isolation of the raw material (xylan frombeech), the evaluation of the transformation process of xylan into thefinal product comprising the steps of depolymerization and sulfation,the isolation and purification procedures of the resultant pentosanpolysulfate, the assessment of production batches of raw material,intermediate and final product.

Then, also these structural features related to the quantification ofthe structural unit will be taken into account for the production andmarketing of pentosan polysulfate for pharmaceutical use; furthermorethese structural features also have to be considered as part of theregistration process of the product before the regulatory authorities,such as for example the FDA, for the authorization of generic versionsof pentosan polysulfate.

The identification of the characteristic structural units of xylan andpentosan polysulfate was carried out as previously described by usingadvanced spectroscopic techniques, however, the methods of the presentinvention are not limited to these techniques, but also include the useof the information relating to the presence and quantification of suchcharacteristic structural unit determined by any analytical techniquesuch as IR spectroscopy, HPLC analysis, analysis of the products ofdepolymerization, ELISA, capillary electrophoresis.

The absence or the presence and the quantification of the signals of thecharacteristic structural unit are determined by any method that allowsthe identification in the preparation of xylan or xylan sulfate as such,their fractions or their hydrolysis products. For example, one or moreof the following methods may be used: nuclear magnetic resonance (NMR),mass spectrometry, for example matrix-assisted laser desorptionionization mass spectrometry (MALDI-MS), electrospray ionization massspectrometry (ESI MS), coupled or not to chromatographic separationtechniques (for example size exclusion, ionic, etc.).

The methods of the present invention preferably use NMR spectroscopy,even more preferably the HSQC NMR spectroscopy.

The use of the information relating to the presence and thequantification of the characteristic structural units for thequalification and the selection of batches of pharmaceutical pentosanpolysulfate active ingredient is of particular importance. In that case,on the basis of this information the decision whether to approve,reject, or possibly rework batches of pentosan polysulfate forpharmaceutical use may be taken.

In order to better illustrate the present invention without limiting it,the following examples are now given.

Example 1

Procedure for the Desulfation of Pentosan Polysulfate

500 mg of pentosan polysulfate (Elmiron®) were converted into the acidform by treatment with an ion exchange resin (Amberlite IR 120 H+) andthe resultant solution (50 mL) was neutralized with pyridine and thenlyophilized. The pyridine salt was dissolved in a solution of DMSO (2mL) containing 10% of H₂O and the solution was heated at 80° C. for 4hours. After this time the solution was added to a volume of water atleast 3 times higher and cooled. The xylan sample without sulfate groupswas isolated after suitable purification operations (precipitation,dialysis, Size Exclusion Chromatography). This procedure was repeated toprepare at least 3 samples (P5146, P5211 and P5212).

Example 2

Procedure for the Recordal of NMR Spectra

The spectra were recorded with a spectrometer Bruker AVANCE 500 or 600MHz equipped with a cryoprobe.

Sample preparation: about 20 mg were dissolved in 0.6 mL of D₂O (99.9%deuterated water D) or in a mixture D₂O/DMSO (deuterated dimethylsulfoxide) in 1:9 ratio and transferred into a tube for NMR analysis of5 mm. The ¹H-NMR spectra were acquired at a temperature of 303 Kelvinwith pre-saturation of the water signal with an interval of recycle of12 s, for a number of scans from 16 to 24 calibrating the spectrum withrespect to TSP.

Example 3

Identification of Characteristic Parameters of the Structure of PentosanPolysulfate or Xylan without Sulfate Groups Via Two-Dimensional (2D) NMR

The 2D NMR experiments can be homonuclear (for example COSY, TOCSY,etc.) or heteronuclear (HSQC, HSQC-DEP, HMQC, HMBC, etc.). All the 2Dspectra (HSQC, HMBC, COSY, TOCSY and HSQC-TOCSY) were carried out onsamples prepared under the same conditions described for the ¹H-NMRspectrum and acquired at a temperature of 303 Kelvin, calibrating thespectrum with respect to TSP if performed in D₂O solvent and withrespect to the peak of DMSO solvent, if the spectra were carried out inDMSO/D₂O solvent.

Two-dimensional spectra DQF (double-quantum-filter)-COSY and 2D-TOCSYwere acquired using from 16 to 24 scans per set of 2048×512 data pointswith zero filling in F1 (4096×2048), a function “shifted (π/3) squaredcosine” was applied before the Fourier transform. The spectrum HSQC(heteronuclear single-quantum coherence) was obtained in “phasesensitivity enhanced pure-absorption mode” with decoupling duringacquisition. The dimensions of the matrix were of 1024×320 data pointsthat were “zero-filled” at 4096×2048 by applying a cosine squarefunction before the Fourier transform.

A HMBC experiment on a xylan sample without sulfate groups showed a longrange correlation between the signal of the residue of xylose and acarboxyl group of the acetyl group.

This signal was identified through an experiment COSY and HSQC-TOCSY asthe position 3 of a xylose residue carrying the acetyl group. Theposition 2 of said residue was found to correlate in the HMBC spectrumwith a residue of glucuronic acid highlighting the presence ofglucuronic acid in the position 2 and of the acetyl group in position 3of the same xylose residue. This explained the splitting of the signalsrelating to the glucuronic acid residue in positions 1, 3, 4 and 5 inaddition to the splitting of the signal of the O-Me group.

The two-dimensional experiments described above allowed also to identifythe chemical shifts of the reducing/non-reducing residues and thecharacteristic parameters of the structure as shown in Table 1. The HMBCexperiment on the sample of Elmiron® (pentosan polysulfate) showedsimilar long-range correlations between the signal corresponding to theposition 3 of the acetylated xylose residue and the carboxyl group ofthe acetyl group. The two-dimensional spectra also allowed theassignment of the signals reported in Table 2.

Example 4

Quantification of Characteristic Parameters of the Structure of Xylanwithout Sulfate Groups and Pentosan Polysulfate

The absence or the presence and the quantification of the signals of thecharacteristic structural units were determined with 2D-NMR spectra ofxylans without sulfate groups obtained by desulfation as described inexample 1 and with the spectra of pentosan polysulfate and wereassociated with the presence of a signal in the region corresponding toresidues of xylose bearing the acetyl group. It resulted that the acetylgroup was not distributed in an equivalent manner between the positions2 and 3 of the repetitive units forming the polysaccharide structure,but was mainly concentrated in the position 3 of the repetitive xiloseunits.

It also resulted that these acetyl groups were mainly bound to theposition 3 of xylose units bearing also a 4-O-methyl-glucuronic acidunit in position 2. The presence of the signal meant that thecharacteristic structural unit can be detected and quantified byintegration as % in comparison with the total number of present residuesof 4-O-methyl-glucuronic acid.

The following table 3 reports the values of the quantification carriedout on different samples.

TABLE 3 Xylan samples without sulfate groups obtained by desulfation ofPentosan polysulfate pentosan polysulfate Elmiron ® samples Elmiron ®P5093 P5196 P5197 P5146 P5211 P5212 Xylose units 60.5% 54.5% 44.0% 58.8%46.0% 37.5% substituted with 4-O-methyl- glucuronic acid that arebearing also the acetyl group (% Ac)

The quantification of the percentage of the xylose units substitutedwith 4-O-methyl-glucuronic acid that are bearing also the acetyl group(% Ac) was calculated by processing the integrals of the NMR signalsaccording to the formula:

% Ac=∫C3-XylAc/∫ΣC1-Glc

wherein:

∫C3-XylAc=integral of the signal of the position 3 of the xylose bearingthe acetyl group in 3 and the 4-O-methyl-glucuronic acid in 2

∫ΣC1-Glc=integral of the sum of the signals of the position C1 of4-O-methyl-glucuronic acid, bound to xylose with acetyl group and boundto xylose without acetyl group.

The analysis was also carried out on a sample of pentosan polysulfatefor pharmaceutical use from a different manufacturer and bought on theIndian market. The signals of OAc group at 2.3 ppm, of C3-XylAc group at5.25/75.96 ppm and of C1-XylAc group at 5.00/106.73 ppm were notdetected in its NMR spectra.

The NMR analysis of this sample showed the presence of structures whichare not present in pentosan sulfates extracted from the commercialproduct Elmiron®, associated with signals in different regions of thespectrum, and in particular between 5.8/6.2 ppm and 95/115 ppm,highlighting the diagnostic capability of this technique for structuralinvestigation.

Example 5

Mass Analysis of Xylan

The mass spectrometry was applied as an orthogonal technique ofstructural analysis. The xylan sample without sulfate groups obtained asdescribed in example 1 was analyzed via MALDI mass spectrometryfollowing the procedure below. The sample was dissolved in H₂O at aconcentration of approximately 0.5 mg/mL. 1 microliter of the analytesolution was added to 5 microliters of a solution of DHB matrix at aconcentration of 10 mg/mL in 80% EtOH. 1 microliter of the mixture wasplaced on the target and analyzed with a spectrometer Bruker DaltonicsAutoflex MALDI TOF in positive polarity and in reflectron mode.

1) A method for the preparation of polysaccharides of formula

wherein R is H or SO₃Na G is H, SO₃Na or acetyl comprising at least oneidentification or quantification step of the xylose units substitutedwith 4-O-methyl-glucuronic acid and acetylated (G=acetyl). 2) A methodaccording to claim 1 wherein the polysaccharide contains at least 20% ofxylose units substituted with 4-O-methyl-glucuronic acid that areacetylated (G=acetyl). 3) A method according to claim 2 wherein thepolysaccharide contains from 35% to 70% of xylose units substituted with4-O-methyl-glucuronic acid that are acetylated (G=acetyl). 4) A methodaccording to claim 1 wherein said identification or quantification stepis carried out by NMR spectroscopy. 5) A method according to claim 4wherein the identification comprises the detection of one or moresignals at 2.17(1H), 23.89(13C); 176.60(13C); 4.63(1H), 104.17(13C);3.68(1H), 78.41(13C); 5.09(1H), 76.99(13C); 3.78(1H), 79.36(13C);4.10(1H), 65.96(13C); 3.37(1H), 65,95(13C); 5.30(1H), 101.16(13C);3.58(1H), 73.93(13C); 3.80(1H), 75.22(13C); 3.82(1H), 84.46(13C);4.64(1H), 72.67(13C); 3.49(1H), 63.00(13C); 177.00(13C); 2.30(1H),23.37(13C); 176.14(13C); 5.00(1H), 106.73(13C); 3.93(1H), 76.85(13C);5.25(1H), 75.96(13C); 4.14(1H), 76.79(13C); 4.29(1H), 65.60(13C);3.69(1H), 65.60(13C); 5.76(1H), 96.99(13C); 4.31(1H), 77.76(13C);4.42(1H), 80.83(13C); 3.45(1H), 84.04(13C); 3.92(1H), 75.71(13C);3.48(1H), 63.61(13C); 178.44(13C). 6) A method according to claim 4wherein the quantification is calculated according to the formula %Ac=∫C3-XylAc/∫ΣC1-Glc, wherein ∫C3-XylAc=integral of the signal of theposition 3 of the xylose bearing the acetyl group in 3 and the4-O-methyl-glucuronic acid in 2 and ∫ΣC1-Glc=integral of the sum of thesignals of the position C1 of 4-O-methyl-glucuronic acid, bound toxylose with acetyl group and bound to xylose without acetyl group. 7) Amethod according to claim 1 for the preparation of pentosan polysulfate.8) A method according to claim 1 for the preparation of xylan. 9) Amethod according to claim 1 for the qualification of pentosanpolysulfate for pharmaceutical use, raw materials thereof andmanufacturing products thereof.